1. Field of the Invention
This invention relates to a near-infrared microscope used for internal observations and measurements on an organic specimen which is transparent or nearly transparent.
2. Description of Related Art
The field of biological research, is changing it's principal object from conventional, morphogenetic observation to the study of mechanisms that transmit information between cells. In keeping with this, an organic specimen which is greater in thickness (for example, a thickness of 200 to 300 .mu.m or more) than previous ones has been often prepared in order to maintain a state as close to an "in vivo" state as possible. However, cells used as the specimen, which lie near the surface of a living body, suffer damage when they are cut off from the living body, and thus fail to bring about a state close to being "in vivo". Hence, in order to observe and measure the cells in a state close being to "in vivo", a lower cell layer lying at least about 50 .mu.m below the surface of the specimen must be visualized. In a phase-contrast microscope widely used to visualize a transparent or nearly transparent specimen, however, when the thickness of the specimen is 200-300 .mu.m, a phenomenon, referred to as "halo", peculiar to the phase-contrast microscope will occur, and thus it becomes difficult to observe microstructures inside the cells. This fact is disclosed by H. Komatsu, "Foundamentals and Application of Optical Microscopy (3)", Jap. J. Appl. Phys., Vol. 60, No. 10 (1991), pp. 1030-1034. On the other hand, even with a differential interference contrast microscope, observations on the specimen are severely affected by scattering of light attributable to the thickness of the specimen, and hence a visual observation is limited to a depth of about 20-30 .mu.m below the surface of the specimen.
More recently, a technique has been developed in which infrared rays that cause ittle scattering are used in a differential interference contrast microscope to observe the interiors of living cells in a thick layer in combination with a contrast enhanced through image processing. The details of such techniques are set forth in the following citations.
Reference 1: Bert Sakmnann and Brwin Neher, "Single-Channel Recording", Second Edition Plenum, New York, pp. 202-206. PA1 Reference 2: H. Kettenmann and R. Grantyn, "Practical Electrophys-iological Methods", Wiley-Liss, New York, pp. 6-10, (Infrared Videomicroscopy of Living Brain Slices). PA1 Reference 3: H. U. Dodt and W. Zieglgansberger, "Visualizing unstained neurons in living brain slices by infrared DIC-videomicroscopy", Brain Res., 537 (1990), pp. 333-336.
In particular, Reference 3 states that neurons lying about 50-100 .mu.m below the surface of a specimen were observed in 300-.mu.m thick rat brain slices.
The above citations bring out the results of observations on the cells which were obtained by using various filters as near-infrared transmitting filters. Specifically, Reference 1 uses a colored glass filter made by Schott, Germany, (trade name: RG9, its spectral transmittance characteristics are as plotted in the graph of FIG. 1). Reference 2 employs a combination of the RG9 filter made by Schott, identical with that of Reference 1, or an interference filter whose transmittance is maximized at a wavelength of 750 nm and a heat absorbing filter made by Schott (trade name: KG4, its spectral transmittance characteristics are as shown in the graph of FIG. 2). Further, Reference 3 uses an interference filter capable of transmitting light of wavelengths in the 750-1050-nm region. However, any of the near-infrared transmitting filters set forth in these citations is designed to transmit light whose wavelengths range from approximately 700 to 1200 nm, and has the following defects.
First of all, reference is made to the problem of chromatic aberration introduced by an imaging optical system, notably an objective lens, of the microscope. In general, the objective lens of the microscope is corrected for chromatic aberration on the assumption that wavelengths are in the visible region, namely the 450-650-nm region. Also, the level of such correction for chromatic aberration is expressed by the name of "apochromat" or "achromat".
An objective lens at the apochromat level, for example, is known by the disclosure of Japanese Patent Preliminary Publication No. Hei 6-160720. However, when an objective lens that is corrected for chromatic aberration, no matter whether this correction is made at the apochromat or achromat level, is used in the 700-1200nm wavelength region, chromatic aberration is remarkably produced, and thus the imaging performance of the objective lens relative to the interior of the specimen is deteriorated. Consequently, in spite of the fact that near-infrared rays with little scattering are used, the prior art merely allows observations on the cells lying at a depth of about 50-100 .mu.m below the surface of the specimen.
Furthermore, the use of wavelengths in the 700-1200nm region may cause thermal deformation of a microscope body and a rise in temperature of the specimen. Specifically, although a halogen lamp is often used as a light source for illumination, radiation ranging in wavelength from about 900 to 1100 nm in this case has the highest intensity. In recent years, a technique, usually called "patch clamp", has been used in which, by a manipulator widely used in the field of biological microscopy, a minute glass electrode with a diameter of several micrometers is brought into close contact with the surface of a cell membrane to investigate the electrical characteristics of a Ca ion channel of the cell membrane. In this case, however, when a wavelength of 900-1100 nm reaches the specimen, its heat causes damage to the specimen or the condition of a solution for conserving the specimen becomes unstable. Consequently, the problem is encountered that it becomes difficult to bring the glass electrode into close contact with the surface of the cell membrane.